Method for evaluating redox activity of nucleic acid molecule and nucleic acid molecule having redox activity

ABSTRACT

The present invention provides a novel technique by which the redox activity of a nucleic acid molecule can be evaluated. An evaluation method of the present invention includes: a detection step of electrochemically detecting a redox reaction to a substrate, the redox reaction being catalyzed by a nucleic acid molecule to be evaluated, using a device that electrochemically detects a redox reaction; and an evaluation step of evaluating redox activity of the nucleic acid molecule from a result of the detection of the redox reaction. As the device, a device in which a base provided with a detection portion is included, the detection portion includes an electrode system, and the nucleic acid molecule to be evaluated is arranged on the base is used. In the present invention, it is preferred that a plurality of kinds of nucleic acid molecule to be evaluated is arranged on the base, and the plurality of kinds of nucleic acid molecules to be evaluated is evaluated by a single device.

TECHNICAL FIELD

The present invention relates to a method for evaluating redox activityof a nucleic acid molecule and a nucleic acid molecule having redoxactivity.

BACKGROUND ART

It is required to detect a target in various fields such as clinicalmedical care, food, and environment. In the detection of a target,interactions with the target are generally utilized, and among them, amethod using an antibody that specifically binds to a target is widelyused. In this method, for example, a target is bound to an antibodylabeled with oxidoreductase such as peroxidase. Then, a chromogenicreaction is conducted by the enzyme in the labeled antibody using achromogenic substrate, and the development in color is detected. By thedetection of the development in color, analyses of the target such asqualitative analysis and quantitative analysis are performed indirectly.

However, since the antibody is obtained by immunizing animals, it isreally difficult to obtain an antigen specific to a toxic target or alow-molecular-weight target. Hence, recently, a nucleic acid that bindsto a target, i.e., a nucleic acid aptamer (hereinafter, merely referredto as an aptamer) has been focused on. The aptamer can be obtained in atest tube. Therefore, for example, it is possible to obtain aptamers toa toxic target and a low-molecular-weight target. Further, it has beenattempted to use such aptamer as substitute for the antibody indetection of a target in combination with DNAzyme exerting catalyticactivity as in peroxidase. The DNAzyme generally is DNA that exerts acatalytic property of peroxidase by having a guanine-rich structuralmotif, having a G-quadruplex structure, and forming a complex by bindingto hemin.

In the detection of a target, a single-stranded nucleic acid elementobtained by linking a single-stranded aptamer and a single-strandedDNAzyme is specifically utilized (Non-Patent Document 1). Thesingle-stranded nucleic acid element forms a stem structure byself-annealing in the absence of a target, and the DNAzyme cannot formG-quadruplex by the stem structure. Therefore, the DNAzyme in thesingle-stranded nucleic acid element cannot bind to hemin and cannotachieve a catalytic property in the absence of a target. On the otherhand, in the presence of a target, the single-stranded nucleic acidelement releases the stem structure by binding the target to theaptamer. Therefore, in the presence of a target, the DNAzyme in thesingle-stranded nucleic acid element forms G-quadruplex and exerts thecatalytic property by binding to hemin Thus, when a chromogenicsubstrate to redox activity is present together, a chromogenic reactionoccurs in the presence of the target, and a chromogenic reaction doesnot occur in the absence of the target. Therefore, it becomes possibleto analyze the target by detecting the chromogenic reaction.Furthermore, it is not required to label the target, and thus, itbecomes possible to directly detect various targets including alow-molecular-weight target.

As described above, the single-stranded nucleic acid element is requiredto control activity of the DNAzyme by a conformation of the aptamer.Therefore, for example, it is desired to combine DNAzyme whose activitycan be easily controlled according to the sequence of an aptamer to beused.

However, there is only a limited number of DNAzymes that have beenreported. Therefore, when a combination with a predetermined aptamer isdecided, there has no choice but to select from the limited number ofDNAzymes. Thus, there is a limitation in structuring a nucleic acidelement with superior accuracy according to the target. Moreover, inorder to make it possible to perform detection with superiorsensitivity, DNAzyme which highly exerts redox activity is required.

Therefore, it has been attempted to obtain novel DNAzyme. However, inscreening of DNAzyme, there has no choice but to determine each ofactivities of candidate nucleic acid molecules, and the operationthereof is really complicated.

PRIOR ART DOCUMENT Non-Patent Document

Non-Patent Document 1: Teller et al., Anal. Chem., 2009, vol. 81, p.9114-9119

SUMMARY OF INVENTION Problem to be Solved by the Invention

Hence, the present invention is intended to provide a novel technique bywhich redox activity of a nucleic acid molecule can be easily evaluated.

Means for Solving Problem

The evaluation method according to the present invention is a method forevaluating redox activity of a nucleic acid molecule, the methodincluding: a detection step of electrochemically detecting a redoxreaction to a substrate, the redox reaction being catalyzed by at leastone nucleic acid molecule to be evaluated, using a device thatelectrochemically detects a redox reaction; and an evaluation step ofevaluating redox activity of the at least one nucleic acid molecule froma result of the detection of the redox reaction, wherein the deviceincludes a base provided with a detection portion, the detection portionincludes an electrode system, and the nucleic acid molecule to beevaluated is arranged on the base.

The nucleic acid molecule according to the present invention is anucleic acid molecule having redox activity, containing at least onepolynucleotide selected from the group consisting of SEQ ID NOs: 1 to132.

Effects of the Invention

According to the evaluation method of the present invention, thepresence or absence of redox activity of a nucleic acid molecule to beevaluated and the intensity of the redox activity can be evaluatedeasily. Moreover, according to the evaluation method of the presentinvention, a plurality of kinds of nucleic acid molecules can beevaluated simultaneously, for example. Therefore, an intended nucleicacid molecule can be effectively screened. The nucleic acid moleculehaving redox activity is, for example, as mentioned above, used assubstitute for an enzyme such as peroxidase and thus is useful invarious fields such as clinical medical care, food, and environment.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A to 1D are graphs showing electrical signals of DNAzyme underdifferent conditions of pH, the length of a spacer on an electrode, andthe concentration of hydrogen peroxide in Example 1 of the presentinvention. FIG. 1A shows results in the case of pH7.4. FIG. 1B showsresults in the case of pH8.0. FIG. 1C shows results in the case ofpH8.5. FIG. 1D shows results in the case of pH9.0.

FIGS. 2A and 2B are graphs showing electrical signals of DNAzyme inExample 1 of the present invention. FIG. 2A is a graph showingreproducibility in a first-time electrical signal measurement and asecond-time electrical signal measurement by the same microarray. FIG.2B is a graph showing reproducibility in DNAzymes having the same lengthof a spacer.

FIG. 3 shows graphs showing a relationship between an electrical signalvalue and its frequency with respect to each DNAzyme in Example 1 of thepresent invention.

FIGS. 4A to 4C are graphs showing electrical signals of DNAzyme inExample 2 of the present invention. FIG. 4A is a graph showingreproducibility in a first-time electrical signal measurement and asecond-time electrical signal measurement by the same microarray. FIG.4B is a graph showing reproducibility in the second-time electricalsignal measurement and a third-time electrical signal measurement by thesame microarray. FIG. 4C is a graph showing reproducibility in thefirst-time electrical signal measurement and the third-time electricalsignal measurement by the same microarray.

FIGS. 5A to 5D are graphs showing a relationship between an electricalsignal value and its frequency of each DNAzyme in Example 2 of thepresent invention. FIG. 5A shows a result in the case of c-Myc. FIG. 5Bshows a result in the case of SA. FIG. 5C shows a result in the case ofEAD2. FIG. 5D shows a result in the case of TA.

FIG. 6 is a graph showing an electrical signal value of each DNAzyme inExample 3 of the present invention.

DESCRIPTION OF EMBODIMENTS 1. Evaluation Method

The evaluation method according to the present invention is, asmentioned above, a method for evaluating redox activity of a nucleicacid molecule, the method including: a detection step ofelectrochemically detecting a redox reaction to a substrate, the redoxreaction being catalyzed by at least one nucleic acid molecule to beevaluated, using a device that electrochemically detects a redoxreaction; and an evaluation step of evaluating redox activity of the atleast one nucleic acid molecule from a result of the detection of theredox reaction, wherein the device includes a base provided with adetection portion, the detection portion includes an electrode system,and the nucleic acid molecule to be evaluated is arranged on the base.

Only one kind of the nucleic acid molecule to be evaluated may be used,and it is preferred that the nucleic acid molecule to be evaluatedincludes a plurality of kinds of nucleic acid molecules, for example. Inthe latter case, for example, by arranging a plurality of kinds ofnucleic acid molecules on the base, the plurality of kinds of nucleicacid molecules can be evaluated using the single base.

The nucleic acid molecule to be evaluated is, for example, a moleculecontaining a nucleotide residue. The nucleic acid molecule may be, forexample, a molecule composed of only a nucleotide residue or a moleculecontaining a nucleotide residue. Examples of the nucleotide includeribonucleotide, deoxyribonucleotide, and derivatives thereof. Thenucleic acid molecule may contain any one kind of, two or more kinds of,or all of ribonucleotide, deoxyribonucleotide, and derivatives thereof.Specifically, the nucleic acid molecule may be, for example, RNAcontaining ribonucleotide and/or a derivative thereof, DNA containingdeoxyribonucleotide and/or a derivative thereof, or a chimera (DNA/RNA)containing the former and the latter. The nucleic acid molecule may be asingle strand or a double strand and is preferably a single strand. Whenthe nucleic acid molecule is a single strand, examples of the nucleicacid molecule include a single-stranded DNA, a single-stranded RNA, anda single-stranded chimera (DNA/RNA). When the nucleic acid molecule is adouble strand, examples of the nucleic acid molecule include adouble-stranded DNA, a double-stranded RNA, a DNA-RNA double strand, anda double-stranded chimera (DNA/RNA). The length of the nucleic acidmolecule is not particularly limited and is, for example, from 11 to 80bases.

The nucleotide may include, as bases, for example, natural bases(non-artificial bases) and non-natural bases (artificial bases).Examples of the natural bases include A, C, G, T, U, and modified basesthereof. Examples of the modification include methylation, fluorination,amination, and thiation. Examples of the non-natural bases include2′-fluoropyrimidine and 2′-O-methylpyrimidine, and specific examplesthereof include 2′-fluorouracil, 2′-aminouracil, 2′-O-methyluracil, and2-thiouracil. The nucleotide may be, for example, modified nucleotide,and examples of the modified nucleotide include a 2′-methylated-uracilnucleotide residue, a 2′-methylated-cytosine nucleotide residue, a2′-fluorinated-uracil nucleotide residue, a 2′-fluorinated-cytosinenucleotide residue, a 2′-aminated-uracil nucleotide residue, a2′-aminated-cytosine nucleotide residue, a 2′-thiated-uracil nucleotideresidue, and a 2′-thiated-cytosine nucleotide residue. Examples of thenucleic acid molecule include PNA (peptide nucleic acid) and LNA (LockedNucleic Acid).

In the present invention, the redox reaction is only necessary to be areaction in which a transfer of electrons between two substrates isgenerated in a process of generating a product from the substrates, forexample. The kind of the redox reaction is not particularly limited. Theactivity that catalyzes the redox reaction can be, for example, activitythat is the same as an enzyme, and can be, specifically, for example,activity that is the same as peroxidase (hereinafter also referred to asperoxidase-like activity). The peroxidase-like activity can be, forexample, horseradish-derived peroxidase (HRP) activity. When the nucleicacid molecule to be evaluated is DNA and has the redox activity, the DNAcan be referred to as DNA enzyme or DNAzyme, for example. When thenucleic acid molecule is RNA and has the redox activity, the RNA can bereferred to as RNA enzyme or RNAzyme, for example.

In the present invention, the detection portion of the device is onlynecessary to detect an electrical signal that is generated by a redoxreaction that is catalyzed by the nucleic acid molecule to be evaluated.The detection portion includes an electrode system as mentioned above.The electrode system may include a working electrode and a counterelectrode or may include a working electrode, a counter electrode, and areference electrode, for example. The material of each electrode is notparticularly limited, and examples thereof include platinum, silver,gold, and carbon. Examples of the working electrode and the counterelectrode include a platinum electrode, a silver electrode, a goldelectrode, and a carbon electrode. The reference electrode can be, forexample, a silver/silver chloride electrode. The silver/silver chlorideelectrode can be formed by laminating a silver chloride electrode on asilver electrode, for example.

The detection portion can be formed by arranging the electrodes on thesurface of the base, for example. A method for arranging the electrodesis not particularly limited, and a conventionally known method can beemployed, for example. Specific examples of the method include thin-filmforming methods such as an evaporation method, a sputtering method, ascreen printing method, and a plating method. The electrodes may bearranged directly or indirectly on the base, for example. The indirectarrangement can be, for example, an arrangement via other members.

The base is not particularly limited and is, for example, preferably abase having an insulating surface. The base may be, for example, a basecontaining an insulating material or being composed of an insulatingmaterial or a base including an insulating layer that has a surfacecontaining an insulating material or an insulating layer composed of theinsulating material. The insulating material is not particularlylimited, and examples thereof include conventionally known materialssuch as glass, ceramics, insulating plastics, and paper. The insulatingplastic is not particularly limited, and examples thereof include asilicone resin, a polyimide resin, an epoxy resin, and a fluorine resin.

As mentioned above, only one kind of the nucleic acid molecule to beevaluated may be used, it is preferred that the nucleic acid molecule tobe evaluated includes a plurality of kinds of nucleic acid molecules,and specifically, the plurality of kinds of nucleic acid molecules isarranged on the base. The device is, for example, preferably amicroarray obtained by arranging the plurality of nucleic acid moleculeson the base. It is preferred that the plurality of kinds of nucleic acidmolecules is arranged on the base in the matrix state, for example. Inorder to make it possible to detect a redox reaction of the nucleic acidmolecule according to the kind thereof, it is preferred that the devicehas a plurality of detection portions, and a different kind of nucleicacid molecule is arranged in each detection portion, for example.Specifically, the device can be formed by fractionating the surface ofthe base into matrixes, forming an electrode system such as mentionedabove in each fractional region to form detection portions, andarranging the nucleic acid molecule in each detection portion, forexample. Moreover, a device obtained by binding the nucleic acidmolecule to be evaluated to a probe on a commercially availableelectrochemical detection-type system microarray can be used as a devicefor evaluation. The commercially available microarray can be, forexample, CombiMatrix ElectraSense microarray (trade name) (manufacturedby CombiMatrix).

The nucleic acid molecule to be evaluated is only necessary to bearranged on the base as mentioned above and is preferably immobilized onthe base. The nucleic acid molecule may be arranged directly orindirectly on the base, for example. Specifically, the nucleic acidmolecule is, for example, preferably arranged on the detection portionin the base, more preferably arranged on any of the electrodes in thedetection portion, and yet more preferably arranged on the workingelectrode among the electrodes. The nucleic acid molecule may bearranged directly or indirectly on the detection portion or any of theelectrodes, for example. Hereinafter, “arrangement or immobilization ofthe nucleic acid molecule on the base” encompasses the meaning ofarrangement or immobilization on the detection portion in the base orany of the electrodes in the detection portion, unless otherwise shown.

A method for arranging the nucleic acid molecule is not particularlylimited, and a known nucleic acid immobilization method can be employed.The above-described immobilization method can be, for example, a methodfor immobilizing a previously provided nucleic acid molecule on thebase, preferably on the detection portion, more preferably on any of theelectrodes. This method is, for example, a method utilizingphotolithography, and a specific example thereof is shown in U.S. Pat.No. 5,424,186 or the like. Furthermore, the immobilization method canbe, for example, a method for synthesizing a nucleic acid on the base,preferably on the detection portion, more preferably on any of theelectrodes. This method can be, for example, a spot method, and aspecific example thereof is shown in U.S. Pat. No. 5,807,522, JPH10-503841, or the like.

The nucleic acid molecule may be immobilized on the base in any of the5′ end side and the 3′ end side.

The nucleic acid molecule is preferably arranged on the base via alinker, for example. The linker preferably contains a nucleotideresidue, for example. The linker may be composed of only a nucleotideresidue or may contain a nucleotide residue, for example. The nucleotideis the same as mentioned above. The length of the linker is notparticularly limited and is, for example, from 1 to 60 bases, preferablyfrom 6 to 60 bases, more preferably from 20 to 30 bases. The linker isalso referred to as a spacer, for example.

In the device, the nucleic acid molecule to be evaluated may be in thestate of linking with an aptamer, for example. Hereinafter, an objectobtained by linking between the nucleic acid molecule to be evaluatedand the aptamer is referred to as a nucleic acid element. The aptamer isnot particularly limited and is, for example, an aptamer capable ofbinding to a specific target. For example, it is preferred that theaptamer is a nucleic acid molecule containing a nucleotide residue as acomponent. The nucleotide is not particularly limited and is the same asmentioned above. For example, the aptamer capable of binding to thetarget can be produced by a conventionally known SELEX (SystematicEvolution of Ligands by Exponential Enrichment) method or the like. Theaptamer may be, for example, a single strand or a double strand and ispreferably a single strand.

The nucleic acid molecule and the aptamer may be, for example, directlyor indirectly bound to each other. In the latter case, they may be boundto each other via a linker, for example. For example, the 5′ end of oneof the nucleic acid molecule and the aptamer may be linked with the 3′end of the other, the 5′ end of the nucleic acid molecule may be linkedwith the 5′ end of the aptamer, or the 3′ end of the nucleic acidmolecule may be linked with the 3′ end of the aptamer.

When the aptamer is linked with the nucleic acid molecule, it ispreferred that one end of the nucleic acid molecule is bound to thebase, and the other end is bound to the aptamer, for example. Asmentioned above, the base can be bound to the nucleic acid molecule viaa linker, and the nucleic acid molecule can be bound to the aptamer viaa linker, for example. It is preferred that the linkage between thenucleic acid molecule and the aptamer is, for example, a linkage betweena single stranded nucleic acid molecule and a single-stranded aptamer.The nucleic acid element obtained by linking between the nucleic acidmolecule and the aptamer may form a stem structure and/or a loopstructure by self-annealing, for example.

With respect to the evaluation method according to the presentinvention, a first embodiment shows, as an example, a method using adevice in which the nucleic acid molecule to be evaluated is arranged,and a second embodiment shows, as an example, a method using a device inwhich the nucleic acid molecule binding to the aptamer is arranged,i.e., a device in which the nucleic acid element is arranged. Thepresent invention is not limited to these embodiments.

First Embodiment

In the first embodiment of the present invention, a device in which anucleic acid molecule to be evaluated is arranged on the base is used asmentioned above.

First, in the detection step, for example, a redox reaction catalyzed bya nucleic acid molecule is electrochemically detected in the presence ofa substrate. When the nucleic acid molecule has redox activity, aproduct is generated from the substrate by the nucleic acid molecule,for example, and in the process of the generation, electrons aretransferred, for example. This electron transfer can beelectrochemically detected as an electrical signal in a detectionportion of the device by applying a voltage to electrodes, for example.The detection of the electrical signal can be performed by measuringintensity of the electrical signal such as a current, for example.

The substrate can be externally supplied to the nucleic acid molecule inthe device in the detection step, for example.

The substrate is not particularly limited, and examples thereof includehydrogen peroxide, 3,3′,5,5′-Tetramethylbenzidine (TMB),1,2-Phenylenediamine (OPD),2,2′-Azinobis(3-ethylbenzothiazoline-6-sulfonic Acid Ammonium Salt(ABTS), 3,3′-Diaminobenzidine (DAB), 3,3′-DiaminobenzidineTetrahydrochloride Hydrate (DAB4HCl), 3-Amino-9-ethylcarbazole (AEC),4-Chloro-1-naphthol (4C1N), 2,4,6-Tribromo-3-hydroxybenzoic Acid,2,4-Dichlorophenol, 4-Aminoantipyrine, 4-Aminoantipyrine Hydrochloride,and luminol.

In the detection step, porphyrin can be present together besides thesubstrate, for example. Some of known DNAzymes exert superior redoxactivity by forming a complex with porphyrin, for example. Thus, in thepresent invention, for example, porphyrin may be caused to be presenttogether to form a complex with the porphyrin and detect redox activityof the complex.

The porphyrin is not particularly limited, and examples thereof includeunsubstituted porphyrin and a derivative thereof. The derivative can be,for example, metal porphyrin obtained by forming a complex ofsubstituted porphyrin and a metal element. The substituted porphyrin canbe, for example, N-methylmesoporphyrin. The metal porphyrin can be, forexample, hemin that is a ferric (Fe³⁺) complex. The porphyrin is, forexample, preferably the metal porphyrin, more preferably hemin.

In the detection step, conditions under which the redox reaction isperformed are not particularly limited. The pH is, for example, from 7.4to 9.0, preferably from 7.4 to 8.5, more preferably 7.4. It is preferredthat the pH is adjusted using a buffer solution, for example, and abuffer solution such as Tris-HCl having the above-mentioned pH can beused, for example. The substrate is preferably added to the nucleic acidmolecule as a substrate solution by mixing with a buffer solution, forexample. The concentration of the substrate in the substrate solution isnot particularly limited and is, for example, from 10 to 200 mmol/L,preferably from 20 to 100 mmol/L, more preferably from 40 to 60 mmol/L,particularly preferably 50 mmol/L.

Then, based on a result of the detection in the detection step, redoxactivity of the nucleic acid molecule is evaluated in an evaluationstep. In the evaluation step, the presence or absence of redox activityor the intensity of the redox activity may be evaluated, for example. Inthe latter case, for example, relative intensity of the activity can beevaluated relative to redox activity of any nucleic acid molecule, andthe any nucleic acid molecule preferably has redox activity.

Second Embodiment

In the second embodiment of the present invention, as mentioned above, adevice in which a nucleic acid molecule to be evaluated bound to anaptamer is arranged on a base is used. The second embodiment is the sameas the first embodiment unless otherwise indicated.

First, in the detection step, for example, a redox reaction catalyzed bya nucleic acid molecule is electrochemically detected in the presence ofa substrate and a target. It is particularly preferred that thedetection step includes a step of detecting a redox reaction in thepresence of the substrate and the in the absence of the target and astep of detecting a redox reaction in the presence of the substrate andthe target. Any of the detection in the absence of a target and thedetection in the presence of a target may be performed prior to theother. For example, it is preferred that the detection in the absence ofa target is performed prior to the other because the target binding toan aptamer is not required to be released.

The reason why the detection in the absence of a target and thedetection in the presence of a target are performed is as follows. Inthe case where an aptamer capable of binding to a target and a nucleicacid molecule having redox activity are used in detection of a target,it is desired that the nucleic acid molecule having redox activityexerts activity only when the target is present and does not exertactivity when the target is absent. This is because, when activity isexerted in the absence of a target, a false-positive result is obtainedin the detection of the target. Therefore, the presence or absence ofredox activity in the absence of a target and the presence or absence ofredox activity in the presence of a target are important forestablishing an electrochemical method in the detection of a targetusing an aptamer.

It is considered that the following structural relationship is satisfiedbetween the nucleic acid molecule and the aptamer in the case where thenucleic acid molecule does not exert activity in the absence of a targetand exerts activity in the presence of a target, for example. Theaptamer generally changes its conformation by recognizing and binding toa target. Therefore, by a conformation of an aptamer in the absence of atarget, the nucleic acid molecule binding to the aptamer has a structurein which activity is switched off. On the other hand, by a conformationof the aptamer changed by binding to the target, suppression of activityof the nucleic acid molecule is released, so that the activity of thenucleic acid molecule is switched on only in the case where a target ispresent.

In the detection step, the substrate and the target can be externallysupplied to the nucleic acid molecule in the device, for example. Theorder of adding the substrate and the target is not particularlylimited. For example, it is possible that one of them is added, and theother is thereafter added, or both of them are added at the same time.Moreover, the porphyrin may be present together as in the firstembodiment.

The substrate is not particularly limited and is the same as mentionedabove. The target and the aptamer are not particularly limited, and adesired target and an aptamer capable of binding thereto can be used.

Then, in the evaluation step, based on a result of the detection in thedetection step, redox activity of the nucleic acid molecule isevaluated. In the evaluation step, the presence or absence of redoxactivity or the intensity of the redox activity may be evaluated, forexample. In the latter case, for example, relative intensity of theactivity can be evaluated relative to redox activity of any nucleic acidmolecule, and the any nucleic acid molecule preferably has redoxactivity.

It is preferred that in the evaluation step, redox activity of a nucleicacid molecule that does not exert redox activity in the absence of atarget is evaluated in the presence of a target, for example. In theevaluation step, the presence or absence of redox activity or theintensity of redox activity may be evaluated, for example. In the lattercase, for example, relative intensity of the activity can be evaluatedrelative to redox activity of any nucleic acid molecule.

2. Screening Method

The screening method according to the present invention is, as mentionedabove, a method for screening a nucleic acid molecule having redoxactivity, including: evaluating redox activity of at least one nucleicacid molecule to be evaluated by the method according to the presentinvention using a device; and screening a nucleic acid molecule havingredox activity.

The screening method according to the present invention is characterizedin that redox activity of the nucleic acid molecule to be evaluated isevaluated by the evaluation method according to the present invention,and other steps and conditions are not at all limited.

In the present invention, based on a result of the evaluation of redoxactivity, a nucleic acid molecule exerting redox activity can beselected, and furthermore, a nucleic acid molecule exerting relativelyintense redox activity can be selected as an intended nucleic acidmolecule, for example. Moreover, as mentioned above, for example, anucleic acid molecule that does not exert activity in the absence of atarget and exerts activity in the presence of a target can be selectedas an intended nucleic acid molecule.

3. Nucleic Acid Molecule Having Redox Activity

The nucleic acid molecule having redox activity according to the presentinvention contains at least one polynucleotide selected from the groupconsisting of the following (a) to (d):

(a) a polynucleotide composed of any of base sequences of SEQ ID NOs: 1to 132;

(b) a polynucleotide being composed of a base sequence obtained bydeletion, substitution, insertion and/or addition of one or more basesin any of the base sequences in the polynucleotide (a) and having redoxactivity;

(c) a polynucleotide being composed of a base sequence having anidentity of 80% or more to any of the base sequences in thepolynucleotide (a) and having redox activity; and

(d) a polynucleotide being composed of a base sequence complementary toa polynucleotide that can hybridize with the polynucleotide (a) understringent conditions and having redox activity.

The nucleic acid molecule according to the present invention may be, forexample, a molecule being composed of or containing any of thepolynucleotides (a) to (d). In the latter case, the nucleic acidmolecule according to the present invention may contain two or more ofthe polynucleotides (a) to (d) as described below, for example. The twoor more of the polynucleotides may be the same sequences or differentsequences. In the latter case, the nucleic acid molecule according tothe present invention may further have a linker and/or an additionalsequence, for example. The nucleic acid molecule according to thepresent invention is also referred to as DNAzyme.

The polynucleotide (a) is a polynucleotide composed of any of basesequences of SEQ ID NOs: 1 to 132.

TABLE 1 SEQ ID NO: Name Sequence 1 c0984 TGAGGGCCGGGTGGGTCGGGAA 2 c0568TGAGGGGAGGGCGGGTCGGGAA 3 c0067 TGAGGGATGGGAGGGAGGGGAA 4 c0192TGAGGGAGGGGCGGGCCGGGAA 5 c0524 TGAGGGGAGGGAGGGGCGGGAA 6 c0451TGAGGGTCGGGAGGGAGGGGAA 7 c0541 TGAGGGGAGGGTGGGCAGGGAA 8 c0629TGAGGGGTGGGCGGGTAGGGAA 9 c0184 TGAGGGAGGGGCGGGTCGGGAA 10 c0760TGAGGGGCGGGCGGGTCGGGAA 11 c0728 TGAGGGGCGGGTGGGTCGGGAA 12 e0064CTGGGCGGGCGGGCGGGA 13 c0719 TGAGGGGCGGGAGGGCGGGGAA 14 c0531TGAGGGGAGGGTGGGAGGGGAA 15 c0711 TGAGGGGCGGGAGGGTGGGGAA 16 c0096TGAGGGATGGGTGGGCCGGGAA 17 c0595 TGAGGGGTGGGTGGGAGGGGAA 18 c0335TGAGGGTTGGGAGGGCGGGGAA 19 c0456 TGAGGGTCGGGAGGGTCGGGAA 20 c0756TGAGGGGCGGGCGGGACGGGAA 21 c0717 TGAGGGGCGGGAGGGCAGGGAA 22 c0712TGAGGGGCGGGAGGGTCGGGAA 23 c0562 TGAGGGGAGGGCGGGATGGGAA 24 c0713TGAGGGGCGGGAGGGGAGGGAA 25 c0735 TGAGGGGCGGGTGGGCGGGGAA 26 e0021CTGGGTGGGTGGGAGGGA 27 c0607 TGAGGGGTGGGTGGGCGGGGAA 28 c0722TGAGGGGCGGGTGGGATGGGAA 29 c0600 TGAGGGGTGGGTGGGTCGGGAA 30 e0032CTGGGTGGGCGGGCGGGA 31 c0544 TGAGGGGAGGGTGGGCCGGGAA 32 c0455TGAGGGTCGGGAGGGTGGGGAA 33 c0605 TGAGGGGTGGGTGGGCAGGGAA 34 c0718TGAGGGGCGGGAGGGCTGGGAA 35 c0586 TGAGGGGTGGGAGGGGTGGGAA 36 c0344TGAGGGTTGGGTGGGTCGGGAA 37 c0152 TGAGGGAGGGGTGGGTCGGGAA 38 c0630TGAGGGGTGGGCGGGTTGGGAA 39 c0632 TGAGGGGTGGGCGGGTCGGGAA 40 c0626TGAGGGGTGGGCGGGATGGGAA

TABLE 2 SEQ ID NO: Name Sequence 41 c0762 TGAGGGGCGGGCGGGGTGGGAA 42c0707 TGAGGGGCGGGAGGGAGGGGAA 43 c0759 TGAGGGGCGGGCGGGTGGGGAA 44 c0543TGAGGGGAGGGTGGGCGGGGAA 45 c0637 TGAGGGGTGGGCGGGCAGGGAA 46 c0606TGAGGGGTGGGTGGGCTGGGAA 47 c0895 TGAGGGCTGGGCGGGCGGGGAA 48 c0753TGAGGGGCGGGCGGGAAGGGAA 49 c0625 TGAGGGGTGGGCGGGAAGGGAA 50 c0584TGAGGGGTGGGAGGGTCGGGAA 51 c0567 TGAGGGGAGGGCGGGTGGGGAA 52 c0599TGAGGGGTGGGTGGGTGGGGAA 53 c0520 TGAGGGGAGGGAGGGTCGGGAA 54 c0636TGAGGGGTGGGCGGGGCGGGAA 55 c0627 TGAGGGGTGGGCGGGAGGGGAA 56 c0519TGAGGGGAGGGAGGGTGGGGAA 57 c0343 TGAGGGTTGGGTGGGTGGGGAA 58 c0628TGAGGGGTGGGCGGGACGGGAA 59 c0383 TGAGGGTTGGGCGGGCGGGGAA 60 c0856TGAGGGCTGGGTGGGTCGGGAA 61 c0709 TGAGGGGCGGGAGGGTAGGGAA 62 c0574TGAGGGGAGGGCGGGCTGGGAA 63 c0842 TGAGGGCTGGGAGGGGTGGGAA 64 c0638TGAGGGGTGGGCGGGCTGGGAA 65 c0583 TGAGGGGTGGGAGGGTGGGGAA 66 c0472TGAGGGTCGGGTGGGTCGGGAA 67 c0463 TGAGGGTCGGGAGGGCGGGGAA 68 e0018CTGGGTGGGAGGGTGGGA 69 c0736 TGAGGGGCGGGTGGGCCGGGAA 70 c0604TGAGGGGTGGGTGGGGCGGGAA 71 c0591 TGAGGGGTGGGAGGGCGGGGAA 72 c0792TGAGGGCAGGGTGGGTCGGGAA 73 c0515 TGAGGGGAGGGAGGGAGGGGAA 74 c0564TGAGGGGAGGGCGGGACGGGAA 75 c0199 TGAGGGACGGGAGGGTGGGGAA 76 c0579TGAGGGGTGGGAGGGAGGGGAA 77 c0714 TGAGGGGCGGGAGGGGTGGGAA 78 c0967TGAGGGCCGGGAGGGTGGGGAA 79 c0727 TGAGGGGCGGGTGGGTGGGGAA 80 c0328TGAGGGTTGGGAGGGTCGGGAA

TABLE 3 SEQ ID NO: Name Sequence 81 c0499 TGAGGGTCGGGCGGGAGGGGAA 82c0708 TGAGGGGCGGGAGGGACGGGAA 83 c0608 TGAGGGGTGGGTGGGCCGGGAA 84 c0535TGAGGGGAGGGTGGGTGGGGAA 85 c0596 TGAGGGGTGGGTGGGACGGGAA 86 e0024CTGGGTGGGTGGGCGGGA 87 c0160 TGAGGGAGGGGTGGGCCGGGAA 88 c0710TGAGGGGCGGGAGGGTTGGGAA 89 c0592 TGAGGGGTGGGAGGGCCGGGAA 90 c0706TGAGGGGCGGGAGGGATGGGAA 91 c0528 TGAGGGGAGGGAGGGCCGGGAA 92 c0563TGAGGGGAGGGCGGGAGGGGAA 93 c0723 TGAGGGGCGGGTGGGAGGGGAA 94 c0211TGAGGGACGGGTGGGAGGGGAA 95 c0179 TGAGGGAGGGGCGGGAGGGGAA 96 c0634TGAGGGGTGGGCGGGGTGGGAA 97 c0588 TGAGGGGTGGGAGGGGCGGGAA 98 c0467TGAGGGTCGGGTGGGAGGGGAA 99 c0589 TGAGGGGTGGGAGGGCAGGGAA 100 c0716TGAGGGGCGGGAGGGGCGGGAA 101 c0522 TGAGGGGAGGGAGGGGTGGGAA 102 c0724TGAGGGGCGGGTGGGACGGGAA 103 c0516 TGAGGGGAGGGAGGGACGGGAA 104 c0582TGAGGGGTGGGAGGGTTGGGAA 105 c0580 TGAGGGGTGGGAGGGACGGGAA 106 c0581TGAGGGGTGGGAGGGTAGGGAA 107 c0590 TGAGGGGTGGGAGGGCTGGGAA 108 c0527TGAGGGGAGGGAGGGCGGGGAA 109 c0532 TGAGGGGAGGGTGGGACGGGAA 110 e0013CTGGGAGGGCGGGAGGGA 111 e0052 CTGGGCGGGAGGGCGGGA 112 c0730TGAGGGGCGGGTGGGGTGGGAA 113 c0521 TGAGGGGAGGGAGGGGAGGGAA 114 e0050CTGGGCGGGAGGGTGGGA 115 c0540 TGAGGGGAGGGTGGGGCGGGAA 116 c0915TGAGGGCGGGGTGGGAGGGGAA 117 c0570 TGAGGGGAGGGCGGGGTGGGAA 118 c0734TGAGGGGCGGGTGGGCTGGGAA 119 t1011 GGGTGGGAAGGGAGG 120 c0764TGAGGGGCGGGCGGGGCGGGAA

TABLE 4 SEQ ID NO: Name Sequence 123 t1113 GGGAGGGACGGGAGG 124 c0900TGAGGGCGGGGAGGGACGGGAA 125 c0899 TGAGGGCGGGGAGGGAGGGGAA 126 c0766TGAGGGGCGGGCGGGCTGGGAA 127 c0573 TGAGGGGAGGGCGGGCAGGGAA 128 t0420GGGCGGGAGGGAGGG 129 t1102 GGGAGGAAGGGTGGG 130 c0602TGAGGGGTGGGTGGGGTGGGAA 131 c0585 TGAGGGGTGGGAGGGGAGGGAA 132 c0536TGAGGGGAGGGTGGGTCGGGAA

In the polynucleotide (b), “one or more” is not limited as long as thepolynucleotide (b) is in a range of having redox activity, for example.The “one or more” is, for example, from 1 to 5, preferably from 1 to 3,more preferably from 1 or 2 in any of the base sequences in thepolynucleotide (a).

In the polynucleotide (c), “an identity” is not limited as long as thepolynucleotide (c) is in a range of having redox activity, for example.The identity is, for example, 80% or more, 85% or more, preferably 90%or more, more preferably 95% or more, 96% or more, 97% or more, yet morepreferably 98% or more, particularly preferably 99% or more. Theidentity can be calculated by an analysis software program such as BLASTor FASTA using default parameters, for example (the same applieshereinafter).

In the polynucleotide (d), “a polynucleotide that can hybridize” is, forexample, a polynucleotide completely or partially complementary to thepolynucleotide (a). The hybridization can be detected by any of varioushybridization assays, for example. The hybridization assays are notparticularly limited, and for example, a method described in Sambrook etal., Molecular Cloning: A Laboratory Manual 2nd Ed., Cold Spring HarborLaboratory Press (1989) or the like can be employed.

In the polynucleotide (d), the “stringent conditions” may be, forexample, any of low stringent conditions, middle stringent conditions,and high stringent conditions. The “low stringent conditions” are, forexample, conditions of 5×SSC, 5×Denhardt's solution, 0.5% SDS, 50%formamide, and 32° C. The “middle stringent conditions” are, forexample, conditions of 5×SSC, 5×Denhardt's solution, 0.5% SDS, 50%formamide, and 42° C. The “high stringent conditions” are, for example,conditions of 5×SSC, 5×Denhardt's solution, 0.5% SDS, 50% formamide, and50° C. Those skilled in the art can set the extent of stringency byappropriately selecting conditions such the temperature, the saltconcentration, the concentration and the length of a probe, the ionicstrength, and the time, for example. As the “stringent conditions”,conditions described in Molecular Cloning: A Laboratory Manual 2nd Ed.,Cold Spring Harbor Laboratory Press (1989) can be employed, for example.

A component of the polynucleotide is, for example, a nucleotide residue.Examples of the nucleic acid molecule according to the present inventioninclude DNA composed of only a deoxyribonucleotide residue and DNAcontaining one or more ribonucleotide residues. In the latter case, the“one or more” is not particularly limited and is, for example, from 1 to3, preferably 1 or 2 in the polynucleotide.

In the nucleic acid molecule according to the present invention, thepolynucleotide is preferably a single-stranded polynucleotide. It ispreferred that the single-stranded polynucleotide is capable of forminga stem structure and a loop structure by self-annealing, for example. Itis preferred that the polynucleotide is capable of forming a stem-loopstructure, an internal loop structure, and/or a bulge structure, forexample.

The nucleic acid molecule according to the present invention may be, forexample, a double-stranded polynucleotide. When the nucleic acidmolecule according to the present invention is a double-strandedpolynucleotide, one of single-stranded polynucleotides of thedouble-stranded polynucleotide is any of the above-describedpolynucleotides, and the other single-stranded polynucleotide is notlimited, for example. The other single-stranded polynucleotide can be,for example, a polynucleotide composed of a base sequence complementaryto any of the above-described polynucleotides. When the nucleic acidmolecule according to the present invention is a double-strandedpolynucleotide, it is preferred that the double-stranded polynucleotideis dissociated into single-stranded polynucleotides by denaturation orthe like prior to the use thereof, for example. The dissociatedsingle-stranded polynucleotides may form a stem structure and a loopstructure as mentioned above, for example.

In the present invention, “being capable of forming a stem structure anda loop structure” includes: actually forming a stem structure and a loopstructure; and being capable of forming a stem structure and a loopstructure even if they are not formed, for example. “Bing capable offorming a stem structure and a loop structure” includes the case wherethe formation is checked experimentally and the case where the formationis predicted by a simulation using a computer or the like, for example.

The nucleic acid molecule according to the present invention has redoxactivity, so that it can be used as a substitute for oxidoreductase andis applicable to detection of a target using an aptamer such asmentioned above, for example.

EXAMPLES Example 1 A. Consideration of Conditions

The conditions under which a redox reaction of a nucleic acid moleculeto be evaluated was detected were considered.

(1) Immobilization of Polynucleotide

A known polynucleotide was immobilized, via a spacer, on an electrode ofa commercially available electrochemical detection-type microarray(trade name: CombiMatrix ElectraSense microarray, manufactured byCombiMatrix) as shown below.

In the known polynucleotide, EAD2 (SEQ ID NO: 133) (see Cheng, X., etal., (2009), Biochemistry, 48, 7817-7823) was used as DNAzyme. As anegative control, DNA aptamer SA (SEQ ID NO: 134) that binds tostreptavidin was used.

EAD2 (SEQ ID NO: 133) CTGGGAGGGAGGGAGGGA SA (SEQ ID NO: 134)CCGACGCACCGATCGCAGGTTCGG

The lengths of the spacer were 0-mer (no spacer), 8-mer, 16-mer, and24-mer. The sequence of the spacer was poly dT.

The immobilization was performed by binding the 3′ end of the spacerwith one electrode of the microarray and binding the 3′ end of the knownpolynucleotide to the 5′ end of the spacer. 250 each of four kinds ofEAD2s (dT0, dT8, dT16, dT24) having different lengths of the spacer andfour kinds of SAs (dT0, dT8, dT16, dT24) having different lengths of thespacer were randomly immobilized on the microarray.

(2) Detection of Redox Reaction

Hydrogen peroxide was added to the microarray as a substrate, and anelectrical signal generated by a redox reaction was measured as acurrent. In the measurement, a measurement device (product name:ElectraSense Reader, manufactured by CombiMatrix) was used (hereinafterthe same). Hydrogen peroxide was added to various buffer solutions so asto have predetermined concentrations (0, 1, 2, 4, 8 mmol/L), and theresultant solutions were added as substrate solutions. As the buffersolutions, a tris buffer solution (pH7.4), a tris buffer solution(pH8.0), a tris buffer solution (pH8.5), and a tris buffer solution(pH9.0) were used, and the pHs of the resultant solutions were adjustedso as to have each pH in parentheses after the addition of hydrogenperoxide.

Results of these are shown in FIGS. 1A to 1D. FIGS. 1A to 1D are graphsshowing electrical signals under each condition. FIG. 1A shows resultsin the case of pH7.4, FIG. 1B shows results in the case of pH8.0, FIG.1C shows results in the case of pH8.5, and FIG. 1D shows results in thecase of pH9.0. Each of FIGS. 1A to 1D shows results under differentconditions of the concentration of hydrogen peroxide and the length ofthe spacer. In each of FIGS. 1A to 1D, the vertical axis indicates asignal ratio (S/BG) of the electrical signal (S) of EAD2 and theelectrical signal (background: BG) of the negative control. As shown inFIG. 1A, the electrical signal reached maximum under the conditions ofpH7.4, the concentration of hydrogen peroxide of 2 mmol/L, and thelength of the spacer of 24-mer.

B. Reproducibility

Redox reactions of 250 each of four kinds of EAD2s (dT0, dT8, dT16,dT24) having different lengths of the spacer were measured under thesame conditions as in “A. Consideration of conditions”.

Results of these are shown in FIGS. 2A and 2B. FIG. 2A is a graphobtained by plotting first-time measurement values and second-timemeasurement values of the EAD2s and the SAs. The measurement values aresignal measurement values that are obtained using the measurement deviceand correspond to currents. (hereinafter the same). The horizontal axisindicates the first-time signal measurement values, and the verticalaxis indicates the second-time signal measurement values. FIG. 2B is agraph showing the logarithm of signal values of each EAD2 by the lengthof the spacer. The graph of FIG. 2A also shows a result of a test of nocorrelation of Pearson's correlation coefficient. In FIG. 2A, acorrelation coefficient R=0.9981756. In FIG. 2B, the standard deviationsof the respective lengths of the spacer were 0.09954021 in the case ofdT0, 0.09435911 in the case of dT8, 0.08528754 in the case of dT16, and0.1027817 in the case of dT24. As shown in FIGS. 2A and 2B, the resultsobtained by the measurements of 250 each of four kinds of EAD2s showedreally high reproducibility.

C. Normality

Whether or not the results obtained by the measurements of 250 each offour kinds of EAD2s (dT0, dT8, dT16, dT24) having different lengths ofthe spacer and the results obtained by the measurements of 250 each offour kinds of SAs (dT0, dT8, dT16, dT24) having different lengths of thespacer in “A. Consideration of conditions” were normally-distributed waschecked. In addition, a P value was determined by a Kolmoforov-Smirnovtest.

Results of these are shown in FIG. 3. FIG. 3 shows graphs showing arelationship between a signal value and its frequency with respect toeach EAD2 and each SA. It was determined that signals of 250 each ofEAD2s and SAs were normally distributed as shown in FIG. 3.

Example 2

A plurality of polynucleotides composed of different sequences wereimmobilized on a microarray chip, and redox reactions thereof weremeasured.

A. Reproducibility

As shown below, a plurality of polynucleotides were immobilized, via aspacer composed of 24-mer poly dT, on an electrode of a commerciallyavailable microarray chip (trade name: CustomArray (registeredtrademark) 12K, manufactured by CombiMatrix).

In the polynucleotides, as DNAzyme, EAD2 (SEQ ID NO: 133), an aptamer SA(SEQ ID NO: 134), c-Myc (SEQ ID NO: 135) that is a partial sequence in apromoter region of a transcription factor c-Myc gene, and TA (SEQ ID NO:136) that is an aptamer to thrombin were used.

EAD2 (SEQ ID NO: 133) CTGGGAGGGAGGGAGGGA SA (SEQ ID NO: 134)CCGACGCACCGATCGCAGGTTCGG c-Myc (SEQ ID NO: 135) TGAGGGTGGGGAGGGTGGGGAATA (SEQ ID NO: 136) GGTTGGTGTGGTTGG

Moreover, modified polynucleotides obtained by modifying sequences ofthe EAD2 (SEQ ID NO: 133), the c-Myc (SEQ ID NO: 135), and the TA (SEQID NO: 136) also were used in the same manner as described above. 64kinds of modified EAD2 obtained by modifying the EAD2, 1024 kinds ofmodified c-Myc obtained by modifying the c-Myc, and 1232 kinds ofmodified TA obtained by modifying the TA were provided.

The immobilization was performed by binding the 3′ end of the spacerwith an electrode of one microarray and binding the 3′ end of the knownpolynucleotide or the modified polynucleotide with the 5′ end of thespacer. 100 each of the EAD2, the c-Myc, and the TA as controls of redoxactivity and 5 each of the modified polynucleotides were randomlyimmobilized on the microarray chip.

Then, redox reactions were measured three times under the sameconditions using the same microarray chip. Specifically, a tris buffersolution (pH7.4) containing 2 mmol/L hydrogen peroxide was added to themicroarray chip, and electrical signals generated by the redox reactionswere measured as currents.

Results of these are shown in FIGS. 4A to 4C. In FIGS. 4A to 4C, FIG. 4Ais a graph obtained by plotting first-time measurement values andsecond-time measurement values. FIG. 4B is a graph obtained by plottingthe second-time measurement values and third-time measurement values.FIG. 4C is a graph obtained by plotting the first-time measurementvalues and the third-time measurement values. Each of the graphs alsoshows a result of a test of no correlation of Pearson's correlationcoefficient. As shown in FIGS. 4A to 4C, the results showed really highreproducibility in the same array.

B. Normality

In graphs of FIGS. 5A to 5D, based on the results obtained by themeasurement in “A. Reproducibility”, FIG. 5A shows a relationshipbetween a signal value and its frequency of 100 c-Mycs, FIG. 5B shows arelationship between a signal value and its frequency of 100 SAs, FIG.5C shows a relationship between a signal value and its frequency of 100EAD2s, and FIG. 5D shows a relationship between a signal value and itsfrequency of 100 TAs. It was determined that the signals were normallydistributed in each of the graphs.

Example 3

New DNAzyme having high redox activity was screened using the microarraychip of Example 2.

From the result obtained by the three-time measurements in Example 2, amodified polynucleotide exerting high activity compared with EAD2 wasfound. This result is shown in FIG. 6. FIG. 6 is a graph showing signalvalues of SA, EAD2, c-Myc, and TA as controls of redox activity and 15kinds of modified polynucleotides exerting redox activity. As shown inFIG. 6, these modified polynucleotides showed high redox activitycompared with EAD2. It was determined by a T test that the signal valuesof these 15 kinds of modified polynucleotides were significant to EAD2.The sequences of these 15 kinds of modified polynucleotides are shownbelow.

TABLE 5 SEQ ID NO: Name Sequence 15 c0711 TGAGGGGCGGGAGGGTGGGGAA 22c0712 TGAGGGGCGGGAGGGTCGGGAA 30 e0032 CTGGGTGGGCGGGCGGGA 33 c0605TGAGGGGTGGGTGGGCAGGGAA 35 c0586 TGAGGGGTGGGAGGGGTGGGAA 39 c0632TGAGGGGTGGGCGGGTCGGGAA 50 c0584 TGAGGGGTGGGAGGGTCGGGAA 55 c0627TGAGGGGTGGGCGGGAGGGGAA 65 c0583 TGAGGGGTGGGAGGGTGGGGAA 76 c0579TGAGGGGTGGGAGGGAGGGGAA 83 c0608 TGAGGGGTGGGTGGGCCGGGAA 90 c0706TGAGGGGCGGGAGGGATGGGAA 97 c0588 TGAGGGGTGGGAGGGGCGGGAA 105 c0580TGAGGGGTGGGAGGGACGGGAA 108 c0527 TGAGGGGAGGGAGGGCGGGGAA

While the invention has been particularly shown and described withreference to exemplary embodiments thereof, the invention is not limitedto these embodiments. It will be understood by those of ordinary skillin the art that various changes in form and details may be made thereinwithout departing from the spirit and scope of the present invention asdefined by the claims

This application is based upon and claims the benefit of priority fromJapanese patent application No. 2011-148562, filed on Jul. 4, 2011, thedisclosure of which is incorporated herein its entirety by reference.

INDUSTRIAL APPLICABILITY

According to the present invention, the presence or absence or theintensity of redox activity of a nucleic acid molecule to be evaluatedcan be evaluated easily. Moreover, by such evaluation method accordingto the present invention, a plurality of nucleic acid molecules can beevaluated at the same time. Thus, an intended nucleic acid molecule canbe screened efficiently, for example. As mentioned above, for example,the nucleic acid molecule having redox activity can be used as asubstitute for an enzyme such as peroxidase and is thus useful invarious fields such as clinical medical care, food, and environment.

1-8. (canceled)
 9. A nucleic acid molecule having redox activity,comprising at least one polynucleotide selected from the groupconsisting of SEQ ID NOs: 1 to 132.